LAB 2 WRITE-UP

Thermal Cycler Engineering

Our re-design is based upon the Open PCR system originally designed by Josh Perfetto and Tito Jankowski.

System Design

Key Features

Instructions

Protocols

MaterialsFor PCR Protocol

Supplied in the Kit

Volume

PCR Reaction Mix (a.k.a. GoTaq Mix)

Tag DNA Polymerase

enough for exp.

MgCl2, dNTP's, Forward and Reverse Primer

Ammounts are all suppliedd in kit

DNA Samples

50 μL each

Patients' DNA

DNA of Rabbit Eyeball

Negative control

Positive Control

SYBR GREEN1

' '

Supplied by User

Amount

Lab Coat

1 or 2

PCR machine made of steel with top that snaps

must clip shut!

Fluorimeter with built in camera and slides with dots farther apart

12 new glass slides

micropipettes

at least 5 for mistakes

Eppendorf tubes

you will have a whole box, but will need one for each test

PCR Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!

1. Label Eppendorf Tubes with 1-17. Each tube should contain 50 microliters of one of the following substances:

Test Tube

Contents

1

Positive Control

2

Negative Control

3

Patient 1, Replicate 1

4

Patient 1, Replicate 2

5

Patient 1, Replicate 3

6

Patient 2, Replicate 1

7

Patient 2, Replicate 2

8

Patient 2, Replicate 3

9

Patient 3, Replicate 1

10

Patient 3, Replicate 2

11

Patient 3, Replicate 3

12

Patient 4, Replicate 1

13

Patient 4, Replicate 2

14

Patient 4, Replicate 3

15

Patient 5, Replicate 1

16

Patient 5, Replicate 2

17

Patient 5, Replicate 3

2. Use properly labeled micropipette to place 17 samples into 17 properly labeled Eppendorf tubes already containing 50 microliters GoTaq mix. To see contents of GoTaq mix, see materials.
3. Place 17 Eppendorf tubes containing DNA and GoTaq Mix in Open PCR Machine. This PCR machine is made of steel in order to decrease safety hazards, and is able to hold more Eppendorf tubes. Therefore, all 17 Eppendorf tubes fit in this PCR. The top of the PCR machine snaps shut and can be clipped down to be less dangerous.
4. To make up for the more Eppendorf tubes in the PCR machine, each cycle is increased by 2 degrees Celsius and more power is needed to increase temperature, making the Thermal Cycler program:
Stage One: 1 cycle, 97 degrees Celsius for 180 seconds
Stage Two: 35 cycles, 97 degrees Celsius for 30 seconds, 59 degrees for 30 seconds, 74 degrees Celsius for 30 seconds
Stage Three: 74 degrees Celsius for 180 seconds
Final Hold: 6 degrees Celsius
5. When PCR is complete, proceed to DNA Measurement Protocol

DNA Measurement Protocol
BE CAREFUL NOT TO CROSS CONTAMINATE!!

1. With permanent marker, clearly number micropipettes. With permanent marker, number Eppendorf tubes at the top. There should be 17 labeled micropipettes and 17 Eppendorf tubes.
2. Transfer each sample from PCR Eppendorf tubes to labeled (1-17) Eppendorf tubes containing 400 microliters of buffer. Get all of the sample into the Eppendorf tubes.
3. Take specially labeled Eppendorf tube (18) with SYBR GREEN I and using specially labeled micropipette (18) place 2 drops on first two centered drops on fluorimeter slide. These dots are further apart on this fluorimeter slide in order to avoid contamination.
4. Take diluted sample from labeled Eppendorf tubes and place 2 drops on top of sample of SYBR GREEN I drop.
5. Turn light on, place beam so that it is passing through the drop.
6. Take picture with built in camera to avoid variability on smart phone camera settings.
7. Use micropipette properly labeled with black strip for waste to discard sample.
8. Repeat 3-7 for all samples (Eppendorf tubes 1-17), the DNA of rabbit eyeball (Eppendorf tube 19), and scintillation vial.
9. Generate pictures in ImageJ to analyze results.